The present invention relates to a process for the simple production of .kappa.-casein glycomacropeptides having useful physiological activities.
It is known that .kappa.-casein glycomacropeptides are sialilated peptides which are produced from .kappa.-casein of cow's milk by the action of proteolytic enzymes such as rennet and pepsin, and they are present in cheese whey.
Hitherto, some methods were reported for the production of .kappa.-casein glycomacropeptides, for example, a method comprising dissolving .kappa.-casein which is isolated from cow's milk in deionized water, reacting the obtained solution with pepsin, adding trichloroacetic acid to the solution to precipitate a para-.kappa.-casein fraction, dialyzing the obtained supernatant against deionized water for desalting, and freeze-drying the obtained solution (Seo et al., Journal of Food Science 53, 80 (1988)); and a method comprising dissolving the above .kappa.-casein in deionized water, adjusting the pH of the solution to 6.7, reacting the solution with rennet, readjusting the pH to 4.6 to precipitate the para-.kappa.-casein, removing the precipitate, dialyzing the obtained supernatant for desalting, and freeze-drying the obtained solution (Published by Academic Press Company, "Milk Protein", pp 200).
However, these methods are conducted in laboratories and are not suitable for mass production.
On the other hand, since the industrial utilization of .kappa.-casein glycomacropeptides has been previously unknown, a method for mass producing the compounds has not been studied.
Since it was reported that after taking .kappa.-casein glycomacropeptides dogs appetite were reduced (Bulletin of Experimental Biology and Medicine, 96, 889, (1983)), it has been found that the compounds can be utilized as food materials for the prevention of obesity.
Further, since it was found that .kappa.-casein glycomacropeptides were effective to prevent the adhesion of E. coli to cells of the intestines, to prevent infection of influenza virus (Japanese Patent Unexamined Publication No. 63-284133) or to protect the adhesion of tartar to teeth (Japanese Patent Unexamined Publication No. 63-233911), the demand for the production of .kappa.-casein glycomacropeptides on a large industrial scale is expected.
Given such conditions, a process for preparing a .kappa.-casein glycomacropeptide from rennet casein curd whey has been reported (Japanese Patent Unexamined Publication No. 63-284199). Since the reaction of the process may proceed without the trichloroacetic acid of the prior art, the process can be utilized in food fields and in massproduction. However, the method is always associated with the production of large amount of the rennet casein curds which are obtained as a by-product of the rennet casein curd whey. If the rennet casein curds are not utilized, the production cost of .kappa.-casein glycomacropeptides becomes expensive.
The inventors of the present invention found that the molecular weight of .kappa.-casein glycomacropeptide changes sharply at pH 4. Using the property, they found a process for producing the .kappa.-casein glycomacropeptide at a moderate price (Japanese Patent Unexamined Publication No. 276542).
The process for the production of .kappa.-casein glycomacropeptides comprises adjusting the pH to below 4 of a solution of milk starting materials such as cheese whey, whey protein concentrate and cheese whey from which protein has been removed, treating the solution by ultrafiltration with a membrane having a molecular weight cut-off of 10,000-50,000 to obtain the filtrate of the solution, preferably readjusting the pH of the filtrate to 4 or higher, and concentrating the obtained filtrate with a membrane having a molecular weight cut-off of 50,000 or less to obtain the desalted concentrate.
According to the above invention, the pH value of the solution of milk starting materials such as cheese whey, whey protein concentrate and cheese whey from which protein has been removed is adjusted. Such a simple operation can provide a process for producing .kappa.-casein glycomacropeptides on a large scale and at a low cost. The obtained product has high purity.
Moreover, in a factory where the whey protein concentrates have been produced, new equipment is not required because .kappa.-casein glycomacropeptides can be produced with the ultrafilter or the reverse osmosis hyperfilter used for preparing the whey protein concentrate. The protein fraction from which the .kappa.-casein glycomacropeptides have been removed can be used as the whey protein concentrates.
However, to lower the production cost of .kappa.-casein glycomacropeptides, the whey protein concentrates should be prepared by desalting and drying after neutralizing the whey proteins which are by-products in the process. Further, when the .kappa.-casein glycomacropeptides are recovered from the whey concentrate obtained by ultrafiltration, the permeation flux is lowered by the protein fouling. As a result, the problem is the necessity of long time operation.
In addition, a method for the mass production of a .kappa.-casein glycomacropeptide is disclosed in Japanese Patent Application No. 2-95686. The method comprises heating a solution containing whey proteins, freezing the solution, thawing the frozen solution, concentrating the obtained supernatant to obtain the desalted concentrate of the .kappa.-casein glycomacropeptide. The cost and the operation time are improved by the method, but the purity of the .kappa.-casein glycomacropeptide is not increased.
Furthermore, the inventors of the present invention found that the electric charge of .kappa.-casein glycomacropeptide was highly negative in comparison with the other milk proteins. Using the property, they found a process for separating the .kappa.-casein glycomacropeptide from the other protein admixtures by using a cation exchanger (Japanese Patent Application No. 2-325166).
In the process, a fraction produced as by-products in the process that uses a cation exchanger to produce whey protein isolates (WPI) from cheese whey as raw materials can be used as raw materials to obtain a .kappa.-casein glycomacropeptide. It is advantageous to the cost. When the solution containing the .kappa.-casein glycomacropeptide thus obtained is concentrated and desalinated through ultrafiltration membranes, fouling problem of whey proteins on the membranes are minimized. Since permeation flux of the filtrate is increased, the operation time is shortened in comparison with conventional methods.